Abstract 3125: EGFRvIII translocates to the nucleus and regulates gene transcription with STAT5

Abstract

Abstract Dysregulation of receptor tyrosine kinases is a major contributor to cancer, including glioblastoma, where amplification and mutation of epidermal growth factor receptor (EGFR) is common. The most common mutation is in-frame deletion of exons 2-7, known as EGFRvIII, EGFR* or ΔEGFR. EGFRvIII confers enhanced tumorigenicity on glioma cells in vivo, reducing apoptosis and increasing proliferation. EGFRvIII signals constitutively and at approximately 5-to-10-fold lower intensity than wild-type. We are investigating the pathways that are preferentially activated by EGFRvIII, using a phosphotyrosine-directed, mass spectrometry-based shotgun phosphoproteomics approach in two different cell lines, LNZ308 (PTEN null) and LN428 (PTEN wt). We identified 433 proteins with 772 peptides out of which 249 proteins corresponding to 354 peptides showed tyrosine phosphorylation. Statistical analysis revealed several signals that were prevalent in EGFRvIII expressing glioma cells, including phosphorylation of STAT5, Gab1 and MIG6. Identification of STAT5 as a target of EGFRvIII prompted an investigation of whether EGFRvIII is active in the nucleus. We have identified EGFRvIII associated with phosphorylated STAT5 on chromatin, and capable of activating the expression of STAT5 target genes, including Aurora A kinase, which positively regulate glioblastoma malignancy. STAT5 promotes expression of the EGFRvIII target gene Bcl-XL, and knockdown of STAT5 reduces the transformation induced by EGFRvIII. Furthermore, we have mutated signals in EGFRvIII that regulate nuclear localization, and are examining the impact of these on its ability to promote tumor growth. In a related study we have used artificial receptor dimerization, mediated by technology from Ariad Pharmaceuticals, to enhance the EGFRvIII signal. A chimeric version of EGFRvIII was created, in which the receptor was fused N-terminally with two FKBP-F36V domains that could be activated by the chemical inducer of dimerization, AP20187. This has resulted in a more oncogenic form of EGFRvIII that emits a stronger signal, and we are using this to deepen our phosphoproteomics analysis to seek additional preferential targets of EGFRvIII action, which will be reported. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3125.